Microscopy and proteomics have both revolutionized our understanding of cell biology: while microscopy provides precise spatial information for small numbers of proteins at a time, proteomic methods can detect thousands of proteins but lack spatial information within cells. It would be transformative to combine the strengths of these two fields, to generate an image of the cell in which the complete molecular composition of every spatial region is defined. Such information would represent a quantum leap in our molecular understanding of cellular function, but is beyond the reach of any current technology. This proposal describes a transformative new technology to bridge proteomics and microscopy, to produce the first spatially-resolved proteomic maps of living cells. The key innovation is a nonspecific labeling enzyme that we can genetically target to any region of interest within live cells. Once targeted, we add a chemical substrate to the cell that is converted by the enzyme into a short-lived and highly reactive molecule that chemically labels any protein in its immediate vicinity. Once tagged, the labeled proteins can be isolated and identified by conventional mass spectrometry. Because we know precisely where in the cell the nonspecific labeling enzyme was targeted (e.g. the synaptic cleft), and because the enzyme-generated reactive molecule has a very small labeling radius, any chemically labeled protein that we detect must reside in the vicinity of the nonspecific enzyme (e.g., in the synaptic cleft in this example). We propose to use this technology to map the complete protein composition of many subcellular regions, focusing particularly on those which are poorly understood at the molecular level - such as the synaptic cleft, the mitochondrial inter-membrane space, and organelle-organelle contact zones. In addition to advancing basic molecular and cell biology, this project has many potential medical applications, such as analysis of patient-derived cells and their responses to therapeutics. Such analysis could shed light on the molecular mechanisms of both disease and drug, using only a small fraction of the cellular material required for current proteomic studies. Just as genomic medicine is now revolutionizing medical care, we envision that this project will open the door to proteomic medicine that will provide a critical new layer of information regarding the function/dysfunction of important biological processes in patients.